The aim of this study was to evaluate the effect of thymol and resveratrol administered
in two different formulation modes (candy and syrup) on the development of Nosema
ceranae and on the longevity of honey bees. Emerging bees from a nosema-free
apiary were individually infected with 1 μL of sucrose syrup containing
18000 spores of N. ceranae, placed in cages, and kept in an incubator at
33 °C and 65% RH. The experimental groups were fed candy or syrup prepared with thymol
(100 ppm) or resveratrol (10 ppm). Infection levels were monitored over a 25 day period by
removal and dissection of two live bees per cage. On day 25, post-infection bees fed with
thymol syrup had significantly lower levels of infection (60 ± 9 million spores/bee)
compared to control bees (138 ± 7 million spores/bee). Bees fed with thymol or resveratrol
syrup lived significantly longer (23 and 25 days, respectively) than bees fed with control
syrup (20 days). Thymol treated syrup appears to be promising in the control of nosema
infection.

1. INTRODUCTION

Nosemosis in European honey bees (Apis mellifera L.) was traditionally
known to be caused by the microsporidian Nosema apis Zander, a fungus. In
recent years, however, Nosema ceranae Fries, originally identified on the
Asian honey bee Apis cerana (Fries et al. , 1996), has been reported to infect European honey bees worldwide (Higes et al. ,
2006; Chauzat et al. , 2007; Huang et al. , 2008; Chen et
al. , 2008) and may be replacing N. apis
(Klee et al. , 2007). Disease caused by
N. apis has always been common throughout the beekeeping world (Matheson,
1996) with reported negative impact on hive
productivity and colony survival over the winter (Fries et al. , 1984; Goodwin et al. , 1990).
N. ceranae causes similar symptoms (Higes et al. , 2006) but, according to some authors, may represent a more serious threat
to apiculture than N. apis and be responsible for the sudden collapses of
bee colonies reported in recent years in many countries (Paxton et al. , 2007; Higes et al. , 2008, 2009). It has recently been shown
that bees infected with N. ceranae have a shortened life span due to
energetic stress and that their feeding behaviour is also affected (Mayack and Naug, 2009; Naug and Gibbs, 2009).

The only known effective substance for the control of nosemosis is the antibiotic
fumagillin (Moffet et al. , 1969), which inhibits the
development of both N. apis (Katznelson and Jamieson, 1952; Liu, 1973) and N.
ceranae (Williams et al. , 2008a). The use
of antibiotics for the treatment of diseased beehives, however, is forbidden in most
European countries. Even where treatments with fumagillin are possible, its use poses the
problem of reoccurrence of the disease (Higes et al. , 2008) as only the vegetative forms of the parasite are killed (MacDonald, 1978; Szabo and Heikel, 1987; Wyborn and McCutcheon, 1987) and the
risk of antibiotic residues in honey is a growing concern to the current honey market
(Martin, 2003). The possibility of controlling nosema
with ecologically-friendly products is therefore much desired throughout the beekeeping
world.

In a previous experiment, in which we evaluated the potential of several natural compounds
in the inhibition of nosema development in caged bees (Maistrello et al. , 2008), both thymol (3-hydroxy-p-cymene)
and resveratrol (3,4’,5-trihydroxystilbene), administered with sugar candy proved promising.
Also in that experiment, no differences were noticed in the appetibility of the treated
candies compared to control candies or in the mortality of non-inoculated bees fed with the
different substances.

Thymol is well known by beekeepers due its suppressive effects against the parasitic mite
Varroa destructor (Chiesa, 1991)
and has been shown not to be toxic to honey bees at the concentrations used in the present
work, either by physical contact (Imdorf et al. , 1995) or per os (Detzel and Wink, 1993;
Ebert et al. , 2007). Furthermore, thymol has been
shown to suppress development of Nosema vespula in Helicoverpa
armigera caterpillars (Rice, 2001) and to
inhibit the growth of pathogenic bacteria and fungi, such as Salmonella
typhimurium, Staphylococcus aureus (Juven et al. , 1994), Aspergillus flavus (Mahmoud,
1999) and Cryptococcus neoformans
(Viollon and Chaumont,1994). Recently,
thymol is being investigated as means of alternative chemical control for several plant
pathogenic fungi (Svircev et al. , 2007; Dambolena et
al. , 2008) as well as human oral bacteria (Bennis et
al. , 2004). Due to its low toxicity (Lenga, 1988) and low residuality in honey (Bogdanov et al. ,
1998), thymol is authorised for use in varroa
control in organic beekeeping, according to EU Regulation 834/2007 on organic production
(EC, 2007). Resveratrol is a natural phytoalexin,
produced by some plants in response to infections caused by phytopathogenic microorganisms,
and is known for its anti-cancer and anti-inflammatory effects in mammals (Luna et al. ,
2009; Pallas et al., 2009; Dann et al. , 2009; Park et
al. , 2009). It has been shown that resveratrol can
inhibit the development of the microsporidian Encephalitazoon cunicoli in
in vitro experiments (Leiro et al. , 2004).

Considering that in the spring colonies are usually fed with syrup rather than candy, in
this experiment our aim was to assess whether the potential nosema control substances,
thymol and resveratrol, were equally effective when administered with syrup compared to
candy, in terms of impact on nosema infection and on the longevity of infected bees.

2. MATERIALS AND METHODS

2.1. Experimental design

Treated candy or syrup were administered to honey bee workers artificially infected with
N. ceranae, and thereafter kept in small wooden, glass-sided cages
measuring 10 × 10 × 20 cm, provided with a small frame and foundation, and gravity feeders
and/or feeding dishes. Each cage contained approximately 30 bees and was incubated at 33
°C and 65% RH in the dark.

2.2. Spore suspension

Bees infected with Nosema ceranae were obtained from an apiary located
in the North-East of Italy where nosema disease is endemic. These bees were crushed in
saline solution, filtered through nylon mesh, and the resulting suspension further
purified by two rounds of centrifugation (800 g, 6 min) and re-suspension in saline
solution. The concentration of N. ceranae spores was then determined by
haemacytometer count (Cantwell, 1970). After another
centrifugation, the spores were re-suspended in the quantity of 50% w/v sucrose solution
necessary to yield a final concentration of 18 000 spores per μL. The
obtained spore suspension was used immediately after preparation. An aliquot of the spore
suspension was used for molecular species identification (Martìn-Hernández et al. , 2007).

2.3. Bees

Newly emerged honey bees (Apis mellifera L., presumably A. m.
ligustica) were obtained from a colony in the CRA-API apiary in Reggio Emilia,
after having analysed a sample of bees from the outer frames of the hive to verify absence
of nosema spores. Combs with emerging bees and capped cells close to emergence were
brought into the laboratory and incubated at 33 °C and 65% RH, in the dark, for 6 hours.
Newly emerged bees were manually collected and individually fed with 1 μL
of the inoculated sucrose syrup suspension before being placed in the cages.

2.4. Treated feed: syrup and candy

Two different modes of administration of the natural compounds were tested: syrup and
candy. A 50% w/v sucrose solution was used to prepare the treated syrups and bee candy was
prepared by mixing icing sugar (85%), honey produced in our apiary (10%), and water (5%).
Ethanol (3,2 μL/g) was used to aid solubility of both thymol and
resveratrol, and added to the control syrup and candy. Previous experiments had shown
that, at the above concentration, the presence of ethanol had not affected either food
consumption or bee mortality. The concentrations of the a.i. in the syrup and candy were
0.1 mg/g of thymol (thymol minimum 99.5%, Sigma) and 0.01 mg/g of resveratrol (resveratrol
approx. 99% GC, Sigma), which were in the same range of the concentrations that had
appeared to be the most promising in the previous experiment. The syrup was administered
to the bees via gravity feeders fitted into each cage, initially containing 4 mL syrup,
which was replaced every 3 days (an up-turned 5 mL plastic syringe from which the point
had been cut off, placed in the cage in such a way that the bees could easily access the
nozzle). The candy contained in a 3 cm diameter Petri dish (9 g) was positioned
upside-down on a 1 cm diameter hole on top of the cage. In the cages where bees were fed
with candy, a gravity feeder containing water was also fitted. The treated feed was
administered to the cages immediately after inoculation. The intake of syrup and candy was
recorded every 3 days.

2.5. Monitoring the infection

Two live bees were removed from each cage at 4 time-points: 8, 13, 19, and 25 days after
inoculation, to monitor the development of the infection. These days were chosen as the
intervals between them correspond to the average length of a N. apis
development cycle (Fries, 1993). The
midgut and rectum of each bee was removed and kept with 1 mL saline solution at –20 °C
until the spore count was performed (Cantwell, 1970). In the cages where all the bees had died by the 25th day the evaluation was
carried out on the most recently dead bees.

Starting from the second day after inoculation the number of dead bees in each cage was
recorded every 2–3 days.

2.6. Statistical analysis

Daily feed intake of bees fed with the differently treated candies or syrups was compared
by means of a two-way analysis of variance (ANOVA), to verify the effect of the two
formulations (syrup and candy) and of the treatments (thymol, resveratrol, and control).

For the calculation of survival times, the live bees collected for monitoring the
infection at 8, 13, 19, and 25 days were considered as censored data. For each differently
fed group (thymol, resveratrol, control, administered via syrup and candy), a life table
was calculated (pooling the data for each group after having individually verified that
there were no statistical differences among the life tables of each individual cage of a
group). For each of the six groups median survival times (the survival time at which the
cumulative survival function is equal to 0.5) were obtained. Paired comparisons were then
carried out with log rank test.

Considering the development of the infection, the mean spore load data of the collected
live bees was log-transformed before analysis due to the skewed distribution.

Statistical analyses examined the differences among the six feeding groups in the four
sampling dates. Comparisons of spore number in honey bee samples collected at each
sampling date from the different feeding groups were made using Kruskal-Wallis
nonparametric test. Mann-Whitney U test was used for the two-samples comparison. A
significance level of α = 0.05 was used to define statistical
differences. A meta-analysis using standardized effect size (Hedges’ g)
followed by two-tailed T-test to obtain the probability value for mean differences was
used to confirm significance of the results. All reported analyses were carried out using
Statistica-StatSoft v. 7.1.

3. RESULTS

Molecular analysis of all the samples showed that spores used in the experiment belonged to
N. ceranae.

3.2. Bee survival

For each of the six groups the survival curves fitted the Gompertz distribution. Results
from survival analysis (Fig. 1) showed that 13 days
after the beginning of the experiment, about 90% of the bees were still alive in all
groups, independently from the kind of formulation or a.i. During the following days,
differences among treatments became apparent, as the bees fed with thymol and resveratrol
treated syrup lived significantly longer than bees fed with control syrup and with treated
and control candy (Tab. I). No differences were
detected in the survival of bees fed with the two a.i. within each formulation mode (Tab.
I). These differences were significant even after
Bonferroni adjustement.

Mean number of spores per bee (× 106) ± SE in honey bees collected at
different sampling dates (d) from each feeding group (4 cages per group, 2 bees per
cage). C = candy, S = syrup. For each formulation mode (candy or syrup), same
letters after mean values ± SE at the end of each column, indicate that values are
not significantly different at P < 0.05 level after
Mann-Whitney U test.

3.3. Development of infection

Spore loads measured on sampled bees on the 1st time point (8 d) were not significantly
different and were very similar in all groups (range 1.32−1.99 × 106
spores/bee), proving that individual inoculation was successful in providing an equal
initial infection level (Tab. II). Development of
the parasite was evident by the 2nd time point (13 d) when differences among the groups
started to appear, although not statistically significant and limited to a 2 × factor. By
the 3rd time point (19 d), differences among groups increased, although not significantly:
the control groups reached infection levels higher than 100 × 106 spores/bee,
while bees fed with the treated formulations had lower spore loads. Differences among the
groups were significant (χ2 = 11.63; P =
0.040) in the last time point (25 d), when bees fed with thymol syrup or candy had the
lowest level of spores.

Within the syrup formulation group on day 25, thymol fed bees contained significantly
fewer spores than both resveratrol (U = 0; P = 0.034) and control (U = 0;
P = 0.021) bees. Within the candy formulation group, the number of
spores in the bees fed with thymol candy was statistically lower than the control group (U
= 0; P = 0.034) and not significantly different from the resveratrol
group (U = 3; P = 0.289), which was not different from control (U = 1;
P = 0.127). These results were confirmed by the comparisons of the
standardized effect sizes (Tab. III). In
cross-comparisons between formulations only thymol syrup versus control candy was
significant (U = 0; P = 0.034).

In bees fed with thymol, development of infection was markedly slower than in the other
groups: the increase of infection between the 3rd and 4th time points was the lowest (only
6% increase in bees fed with thymol syrup), and in thymol candy even a decrease of
infection was observed (–35%). By the end of the experiment, control candy and thymol
syrup had respectively the highest and lowest levels of infection (Tab. II).

4. DISCUSSION

From this experiment it emerges that feed intake did not differ according to treatment of
feed with different a.i., in agreement with findings from a previous experiment (Maistrello
et al. , 2008) in which thymol and resveratrol
treated candies were used. In addition to confirming lack of difference in candy intake,
here we showed that intake of syrup also did not differ according to treatment. However,
intake was significantly different on the basis of formulation, with syrup being consumed
twice as much as candy. This was expected, as the bees fed with candy also had ad libitum
access to water. It must therefore be underlined that although the concentrations of the
a.i. were the same, the actual dose of a.i. differed according to a 2-fold factor in the 2
formulation modes.

Thymol, administered either via syrup or via candy, was clearly able to reduce development
of N. ceranae in the midgut, as deduced by the lower spore counts compared
to the control groups observed from the second time point (13 d) until the last (25 d) when
spore loads were halved. It has been suggested that thymol affects both bacteria (Shapiro
and Guggenheim, 1995) and fungi (Svircev et al. ,
2007; Dambolena et al. , 2008) by causing perforation of plasma membrane leading to extra-cellular
leakage, and in the case of yeast also by disrupting the cell wall (Bennis et al. , 2004). The average daily dose of thymol assumed by each
bee with candy was 3.2 × 10−3 mg: this amount appeared to be sufficient to
partially inhibit development of the infection. The daily dose assumed with syrup was twice
as much but a further reduction of infection was not observed, suggesting absence of
dose-dependent effect and that a threshold level was reached with the dose administered via
candy. In our previous experiment (Maistrello et al. , 2008), however, a slightly higher concentration of thymol (0.12 mg/g) administered
in candy had led to a greater inhibition of nosema development than the one observed in the
current trial. Further research is needed to clarify this aspect.

The presence of thymol or resveratrol in syrup caused nosema-infected bees to live
significantly longer than control bees or bees fed with treated candies. In the case of
thymol, higher survival might be related to the lower spore load, whereas in the case of
resveratrol (where spore loads were not different from control bees), higher survival might
be explained by specific life-prolonging antioxidant properties of this substance. It has
been shown that resveratrol can prolong the lifespan of the invertebrates
Caenorhabditis elegans and Drosophila melanogaster (Wood
et al. , 2004) and of the short-lived fish
Nothobranchius furzeri by activating enzymes that promote cell survival
(Valenzano et al. , 2006). In the budding yeast
Saccharomyces cerevisiae, resveratrol increases DNA stability and extends
cell lifespan by 70% (Howitz et al. , 2003).
Therefore, the higher survival of bees fed with resveratrol in the syrup formulation mode
compared to candy could be explained by the differences in the average daily intake, twice
as much in the case of syrup. Indeed, these data confirm results from our previous work
(Maistrello et al. , 2008) in which bees fed with
resveratrol candy survived longer than control bees notwithstanding the same infection
level.

The median survival time observed in bees fed with non-treated formulations was higher than
that observed by Higes et al. (2007) in an artificial
infection experiment, where total mortality on day 8 post-infection was 100%. However, the
infection dose used in that experiment was almost 10-fold than the one used in the present
trial (125 000 vs. 18 000 spores/bee respectively). Median survival times in A. m.
ligustica bees inoculated with 10 000 N. apis spores each and
fed with sucrose syrup had been observed to be 22 days (Malone and Stefanovic, 1999), similar to the median survival time observed in
the control bees fed with syrup in the present experiment (20 days, bees initially
inoculated with 18 000 N. ceranae spores each). Another possibility for
differences between results of this study and those of Higes et al. (2007) is variability in N. ceranae virulence. It has
been shown that different haplotypes of Nosema spp. in honey bees exist
(Williams et al. , 2008b), although it is not known
whether there is a corresponding difference in virulence. However, in bumble bees, Tay et
al. (2005) showed that different haplotypes of
N. bombi are likely to vary in virulence, so the same may be true for
N. ceranae. It is also possible that Higes et al. ’s (2007) bees were exposed to some other agent which may
have decreased their longevity synergistically with N. ceranae, such as
pesticides or viruses. Otherwise, differences could be explained by the different
susceptibility of the bees used in the experiment, although in the case of N. apis
no differences in race susceptibility were detected in several experiments (Malone
et al. , 1995; Malone and Stefanovic, 1999). However, no information on genetic susceptibility
is yet available for infection with N. ceranae.

Feeding colonies with thymol syrup may represent an efficient way of reducing nosema
infection in the hive, as results from this experiment show that, in laboratory conditions,
bees fed with thymol have lower spore loads and live longer than control bees. Indeed, if a
“wellness”index is calculated as follows: proportion of survivors/spore load, measured on
the last time point (25 d), bees fed with thymol syrup have the highest value (2.4 ×
10−9), followed by the group fed with resveratrol syrup (1.9 ×
10−9), whereas bees fed with control candy (1.9 × 10−10) had a
ten-fold lower value. In future experiments, it may be interesting to combine the nosema
inhibiting effect of thymol and the longevity-extending effect of resveratrol. Furthermore,
the results from laboratory tests need to be evaluated at the colony level.

Acknowledgments

We wish to thank Francesco Leonardi, Giovanna Marani for collaboration in experimental
activities and Franco Mutinelli and Anna Granato for their assistance with molecular
analysis. We also thank beekeeper Angelo Barberis for providing nosema diseased bees, Simone
Franceschetti for the work in the bee yard and a kind friend for encouragement.

EC (2007) Council Regulation N. 834/2007 of 28 June 2007 on organic production and labelling of organic products and repealing Regulation (EEC) N. 2092/91, Official Journal of the European Union L189, 20.07.2007, pp. 1–23.
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Mean number of spores per bee (× 106) ± SE in honey bees collected at
different sampling dates (d) from each feeding group (4 cages per group, 2 bees per
cage). C = candy, S = syrup. For each formulation mode (candy or syrup), same
letters after mean values ± SE at the end of each column, indicate that values are
not significantly different at P < 0.05 level after
Mann-Whitney U test.